Analysis of the expression and antioxidative property of a peroxiredoxin 6 from Scophthalmus maximus

Peroxiredoxins (Prxs) are a group of antioxidant proteins that protect cells from oxidative damage caused by various peroxides. To date, six different isoforms of peroxiredoxin (Prx1 to Prx6) have been identified, of which, Prx6 belongs to the 1-Cys Prx subfamily. Although Prx6 of several fish species have been reported at sequence level, there are very few documented studies on the potential function of fish Prx6. In this report, we describe the identification and analysis of a Prx6 homologue, SmPrx6, from turbot Scophthalmus maximus. The full length cDNA of SmPrx6 contains a 5'- untranslated region (UTR) of 60 bp, an open reading frame of 666 bp, and a 3'-UTR of 244 bp. The deduced amino acid sequence of SmPrx6 shares 81-87% overall identities with known fish Prx6. In silico analysis identified in SmPrx6 a conserved Prx6 catalytic motif, PVCTTE, and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. Expression of SmPrx6 was detected in most fish organs, with the highest expression levels found in blood and heart and the lowest level in spleen. Experimental challenges with bacterial pathogens and poly(I:C) upregulated SmPrx6 expression in liver and spleen in a manner that is dependent on the challenging agent and the tissue type. Treatment of cultured primary hepatocytes with H2O2 enhanced SmPrx6 expression in a dose-dependent manner. Recombinant SmPrx6 expressed in and purified from Escherichia coli exhibited thiol-dependent antioxidant activity and could protect cultured hepatocytes from H2O2-induced oxidative damage. Taken together, these results indicate that SmPrx6 is a Prx6 homologue with antioxidative property and is likely to be involved in both cellular maintenance and protective response during host immune defense against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

Differential regulation of Sciaenops ocellatus viperin expression by intracellular and extracellular bacterial pathogens

Viperin is an antiviral protein that has been found to exist in diverse vertebrate organisms and is involved in innate immunity against the infection of a wide range of viruses. However, it is largely unclear as to the potential role played by viperin in bacterial infection. In this study, we identified the red drum Sciaenops ocellatus viperin gene (SoVip) and analyzed its expression in relation to bacterial challenge. The complete gene of SoVip is 2570 bp in length and contains six exons and five introns. The open reading frame of SoVip is 1065 bp, which is flanked by a 5'-untranslated region (UTR) of 34 bp and a 3'-UTR of 350 bp. The deduced amino acid sequence of SoVip shares extensive identities with the viperins of several fish species and possesses the conserved domain of the radical S-adenosylmethionine superfamily proteins. Expressional analysis showed that constitutive expression of SoVip was relatively high in blood, muscle, brain, spleen, and liver, and low in kidney, gill, and heart. Experimental challenges with poly(I:C) and bacterial pathogens indicated that SoVip expression in liver was significantly upregulated by poly(I:C) and the fish pathogen Edwardsiella tarda but down-regulated by the fish pathogens Listonella anguillarum and Streptococcus iniae. Similar differential induction patterns were also observed at cellular level with primary hepatocytes challenged with E. tarda, L anguillarum, and S. iniae. Infection study showed that all three bacterial pathogens could attach to cultured primary hepatocytes but only E. tarda was able to invade into and survive in hepatocytes. Together these results indicate that SoVip is involved in host immune response during bacterial infection and is differentially regulated at transcription level by different bacterial pathogens. (C) 2010 Elsevier Ltd. All rights reserved.

Dissection and localization of the immunostimulating domain of Edwardsiella tarda FliC

Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn (Fenneropenaeus chinensis)

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048 bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes. (c) 2005 Elsevier Ltd. All rights reserved.

Expression of Scophthalmus maximus CD83 correlates with bacterial infection and antigen stimulation

CD83 is a transmembrane glycoprotein of the immunoglobulin (Ig) superfamily and a surface marker for fully matured dendritic cells (DCs) in humans and mice. In teleosts, DC-like cells and their molecular markers are largely unknown. In this report, we described the identification and expressional analysis of a CD83 homologue, SmCD83, from turbot Scophthalmus maximus. The open reading frame of SmCD83 is 639 bp, which is preceded by a S'-untranslated region (UTR) of 87 bp and followed by a 3'-UTR of 1111 bp. The SmCD83 gene is 4716 bp in length, which contains five exons and four introns. The deduced amino acid sequence of SmCD83 shares 40-50% overall identities with the CD83 of several fish species. Like typical CD83, SmCD83 possesses an Ig-like extracellular domain, a transmembrane domain, and a cytoplasmic domain. The conserved disulfide bond-forming cysteine residues and the N-linked glycosylation sites that are preserved in CD83 are also found in SmCD83. Expressional analysis showed that constitutive expression of SmCD83 was high in gill, blood, spleen, muscle, and kidney and low in heart and liver. Bacterial infection and poly(I:C) treatment enhanced SmCD83 expression in kidney in time-dependent manners. Likewise, bacterial challenge caused significant induction of SmCD83 expression in cultured macrophages. Vaccination of turbot with a bacterin and a purified recombinant subunit vaccine-induced significant SmCD83 expression during the first week following vaccination. These results demonstrate that SmCD83 expression correlates with microbial challenge and antigen stimulation, which suggests the possibility that there may exist in turbot DC-like antigen-presenting cells that express SmCD83 upon activation by antigen uptake. In addition, these results also suggest that SmCD83 may serve as a marker for activated macrophages in turbot. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of the immune effects of CpG motifs that protect Japanese flounder (Paralichthys olivaceus) against bacterial infection

CpG-containing oligodeoxynucleotides (ODNs) are known to be immunostimulatory in vertebrate systems and can activate both innate and adaptive immune responses. In this report, we described the selection, identification, and analysis of CpG motifs with immunoprotective effects in Japanese flounder. Sixteen CpG ODNs were synthesized and examined for the ability to inhibit bacterial dissemination in Japanese flounder blood. Four ODNs with the strongest inhibitory effects were selected and mixed to form ODNs 4M. In addition, a plasmid, pCN6, was constructed that contains the sequences of the four selected ODNs. When administered into Japanese flounder via intraperitoneal injection, both ODNs 4M and pCN6 could, in dose and time dependent manners, afford short-term protection against the infections of two different bacterial pathogens. Immunological analyses showed that ODNs 4M and, especially, pCN6 activated head kidney macrophages and enhanced serum bactericidal activity via probably the alternative pathway of complement activation. When used as a DNA vaccine to immunize Japanese flounder, pCN6 conferred apparent protections (42.9% and 52.6%, respectively, in terms of relative percent survival) against the challenges of two different fish pathogens at 4-week post-vaccination. Transcriptional analysis showed that vaccination with pCN6 upregulated the expression of the genes encoding NKEF, MHC II alpha, IL-1 beta, Mx, and MHC I alpha. These results demonstrate that ODNs 4M and pCN6 are immunostimulatory in Japanese flounder and can induce short- and long-term nonspecific protections against bacterial infections. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and characterization of a Cystatin gene from Chinese mitten crab Eriocheir sinensis

Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Enocheir sinensis (designated EsCystain) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 548 and the predicted molecular weight of 13 39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystain were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0 6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01)at 24 h Afterwards. EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2 8-fold of that in blank (P < 0 01)) The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain When the concentration of EsCystatin protein was of 300 mu g mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms. (C) 2010 Elsevier Ltd All rights reserved.

Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly unregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. (C) 2010 Elsevier Ltd. All rights reserved.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

A Dorsal homolog (FcDorsal) in the Chinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge

Rel/NF kappa B is a family of transcription factors. In the present study, a Rel/NF kappa B family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627 bp, revealed a 1071 bp open reading frame encoding 357 aa. The predicted molecular weight (MW)of the deduced amino acid sequence of FcDorsal was 39.78 kDa, and its theoretical pl was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G(+)) bacteria (Micrococcus lysodeikticus) and Gram-negative (G(-)) bacteria (Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NF kappa B member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a Sciaenops ocellatus ISG15 homologue that is involved in host immune defense against bacterial infection

ISG15 is an interferon-stimulated gene that encodes a ubiquitin-like protein. ISG15 homologues have been identified in a number of fish species, some of which are known to be regulated at expression level by virus infection and lipopolysacchande (LPS) treatment However, the relationship between ISG15 and live bacterial infection has not been investigated in piscine models. In this study, an ISG15 homologue, SoISG15, was identified from red drum Scraeriops ocellaws and analyzed at expression and functional levels The open reading frame ofSolSG15 is 477 base pairs (bp) and mtronless, with a 5'-untranslated region (UTR) of 91 bp and a 3'-UTR of 415 bp The deduced amino acid sequence of S0ISG15 shares 60-67% overall identities with the ISG15 of several fish species. S0ISG15 possesses two conserved ubiquinn-like domains and the canonical ubiquitin conjugation motif, LRGG, at the C-terminus. Expressional analysis showed that constitutive expression of SolSG15 was highest in blood and lowest in kidney Experimental challenges with LPS and bacterial pathogens induced significant S0ISG15 expression in the kidney but not in the liver Similar differential induction was also observed at cellular level with primary hepatocytes and head kidney (HK) lymphocytes. Poly(' C), however, effected drastic induction of S0ISG15 expression in kidney and liver at both tissue and cellular levels. Immunoblot analysis showed that S0ISG15 was secreted by cultured HK lymphocytes into the extracellular milieu. Recombinant S0ISG15 expressed in and purified from Eschenclua colt was able to enhance the respiratory burst activity, acid phosphatase activity, and bactericidal activity of HK macrophages. Taken together, the results of this study indicated that SoISG 15 possesses apparent immunological property and is likely to be involved in host immune defense against bacterial infection. (C)2010 Elsevier Ltd All rights reserved.